Use of colostrinin,constituent peptides thereof, and analogs thereof as modulators of intracellular signaling molecules

ABSTRACT

The present invention provides methods that utilize compositions containing colostrinin, a constituent peptide thereof, an active analog thereof, and combinations thereof, as modulators of intracellular signaling molecules, for example.

[0001] This application claims priority to U.S. Provisional ApplicationSer. No. 60/420,369, filed Oct. 22, 2002, and is a Continuation-In-Partof U.S. patent application Ser. No. 10/281,652, filed on Oct. 28, 2002,which is a Divisional of U.S. patent application Ser. No. 09/641,803,filed Aug. 17, 2000 (issued on Dec. 31, 2002 as U.S. Pat. No.6,500,798), which claims the benefit of U.S. Provisional ApplicationSer. No.60/149,310, filed Aug. 17, 1999, all of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] Colostrum is a component of the milk of mammals during the firstfew days after birth. Colostrum is a thick yellowish fluid and is thefirst lacteal secretion post parturition and contains a highconcentration of immunogloblins (IgG, IgM, and IgA) and a variety ofnon-specific proteins. Colostrum also contains various cells such asgranular and stromal cells, neutrophils, monocyte/macrophages, andlymphocytes. Colostrum also includes growth factors, hormones, andcytokines. Unlike mature breast milk, colostrum contains low sugar, lowiron, but is rich is lipids, proteins, mineral salts, vitamins, andimmunoglobins.

[0003] Colostrum also includes or contains a proline-rich polypeptideaggregate or complex, which is referred to as colostrinin (CLN). Onepeptide fragment of colostrinin is Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro(SEQ ID NO:31), which is disclosed in International Publication No.WO-A-98/14473. Colostrinin and this fragment have been identified asuseful in the treatment of disorders of the central nervous system,neurological disorders, mental disorders, dementia, neurodegenerativediseases, Alzheimer's disease, motor neurone disease, psychosis,neurosis, chronic disorders of the immune system, diseases with abacterial and viral aetiology, and acquired immunological deficiencies,as set forth in International Publication No. WO-A-98/14473.

[0004] Although certain uses for colostrinin have been identified, itwould represent an advancement in the art to discover and disclose otheruses for colostrinin, or a component thereof, that are not readilyascertainable from the information currently known about colostrinin orits constituents.

SUMMARY OF THE INVENTION

[0005] The present invention relates to the use of colostrinin, at leastone constituent (i.e., component) peptide thereof, at least one activeanalog thereof (e.g., peptide having an N-terminal sequence equivalentto an N-terminal sequence of at least one of the colostrinin constituentpeptides), and combinations thereof, as modulators of intracellularsignaling mechanisms. The signaling molecules discovered to date thatare modulated include 4HNE adduct formation, GSH, P53, and JNK.

[0006] Furthermore, the present invention relates to the use ofcolostrinin, at least one constituent (i.e., component) peptide thereof,at least one active analog thereof (e.g., peptide having an N-terminalsequence equivalent to an N-terminal sequence of at least one of thecolostrinin constituent peptides), and combinations thereof, in theinhibition of apoptosis. Specifically, the apoptotic (cytotoxic) effectof B amyloid on SH—SY5Y neuronal cells and TNF-alpha.

[0007] In one embodiment, the present invention provides a method ofmodulating an intracellular signaling molecule in a cell. The methodincludes contacting the cell with a modulator selected from the group ofcolostrinin, a constituent peptide thereof, an active analog thereof,and combinations thereof, under conditions effective to accomplish atleast one of the following: reduce 4HNE-protein adduct formation;inhibit 4HNE-mediated glutathione depletion; inhibit 4HNE-inducedactivation of p53 protein; or inhibit 4HNE-induced activation of c-JunNH2-terminal kinases.

[0008] In one embodiment, the present invention provides a method ofdown regulating 4HNE-mediated lipid peroxidation in a cell. The methodincludes contacting the cell with a modulator selected from the group ofcolostrinin, a constituent peptide thereof, an active analog thereof,and combinations thereof, wherein: the active analog is an active analogof a constituent peptide of colostrinin selected from the group of SEQID NO:1 through SEQ ID NO:34; the active analog comprises a peptidehaving an amino acid sequence with at least about 15 percent proline andhaving at least about 70 percent structural similarity to one or moreconstituent peptides of colostrinin; and the active analog does notinterfere with cellular uptake of redox-sensitive2′,7′-dihydro-dichlorofluorescein-diacetate.

[0009] In one embodiment, the present invention provides a method forinhibiting apoptosis in a cell (typically, due to DNA damage). Themethod includes contacting the cell with an effective amount of anapoptosis inhibitor selected from the group of colostrinin, aconstituent peptide thereof, an active analog thereof, and combinationsthereof.

[0010] In another embodiment of inhibiting apoptosis in a cell, a methodis provided that includes contacting the cell with an effective amountof an apoptosis inhibitor selected from the group of colostrinin, aconstituent peptide thereof, an active analog thereof, and combinationsthereof, wherein; the active analog is an active analog of a constituentpeptide of colostrinin selected from the group of SEQ ID NO:1 throughSEQ ID NO:34; the active analog comprises a peptide having an amino acidsequence with at least about 15 percent proline and having at leastabout 70 percent structural similarity to one or more constituentpeptides of colostrinin; and the active analog does not interfere withcellular uptake of redox-sensitive2′,7′-dihydro-dichlorofluorescein-diacetate.

[0011] Other methods of the present invention include protecting againstDNA damage in a cell, and reducing the toxic effect of P-amyloid orretinoic acid on a cell. These methods involve contacting the cell withan effective amount of a compound selected from the group ofcolostrinin, a constituent peptide thereof, an active analog thereof,and combinations thereof.

[0012] The cell can be present in a cell culture, a tissue, an organ, oran organism. For certain embodiments, the cell is a mammalian cell. Forcertain embodiments, the cell is a human cell.

[0013] For certain embodiments, the compound (e.g. modulator such as anapoptosis inhibitor) is a constituent peptide of colostrinin.Preferably, the modulator is selected from the group of MQPPPLP, (SEQ IDNO:1) LQTPQPLLQVMMEPQGD, (SEQ ID NO:2) DQPPDVEKPDLQPFQVQS, (SEQ ID NO:3)LFFFLPVVNVLP, (SEQ ID NO:4) DLEMPVLPVEPFPFV, (SEQ ID NO:5) MPQNFYKLPQM,(SEQ ID NO:6) VLEMKFPPPPQETVT, (SEQ ID NO:7) LKPFPKLKVEVFPFP, (SEQ IDNO:8) VVMEV, (SEQ ID NO:9) SEQP, (SEQ ID NO:10) DKE, (SEQ ID NO:11)FPPPK, (SEQ ID NO:12) DSQPPV, (SEQ ID NO:13) DPPPPQS, (SEQ ID NO:14)SEEMP, (SEQ ID NO:15) KYKLQPE, (SEQ ID NO:16) VLPPNVG, (SEQ ID NO:17)VYPFTGPIPN, (SEQ ID NO:18) SLPQNILPL, (SEQ ID NO:19) TQTPVVVPPF, (SEQ IDNO:20) LQPEIMGVPKVKETMVPK, (SEQ ID NO:21) HKEMPFPKYPVEPFTESQ, (SEQ IDNO:22) SLTLTDVEKLHLPLPLVQ, (SEQ ID NO:23) SWMHQPP, (SEQ ID NO:24)QPLPPTVMFP, (SEQ ID NO:25) PQSVLS, (SEQ ID NO:26)LSQPKVLPVPQKAVPQRDMPIQ, (SEQ ID NO:27) AFLLYQE, (SEQ ID NO:28) RGPFPILV,(SEQ ID NO:29) ATFNRYQDDHGEEILKSL, (SEQ ID NO:30) VESYVPLFP, (SEQ IDNO:31) FLLYQEPVLGPVR, (SEQ ID NO:32) LNF, (SEQ ID NO:33) andMHQPPQPLPPTVMFP, (SEQ ID NO:34)

[0014] and combinations thereof.

[0015] As used herein, “a” or “an” means one or more (or at least one),such that combinations of active agents (i.e., active oxidative stressregulators), for example, can be used in the compositions and methods ofthe invention. Thus, a composition that includes “a” polypeptide refersto a composition that includes one or more polypeptides.

[0016] “Amino acid” is used herein to refer to a chemical compound withthe general formula: NH₂—CRH—COOH, where R, the side chain, is H or anorganic group. Where R is organic, R can vary and is either polar ornonpolar (i.e., hydrophobic). The amino acids of this invention can benaturally occurring or synthetic (often referred to asnonproteinogenic). As used herein, an organic group is a hydrocarbongroup that is classified as an aliphatic group, a cyclic group orcombination of aliphatic and cyclic groups. The term “aliphatic group”means a saturated or unsaturated linear or branched hydrocarbon group.This term is used to encompass alkyl, alkenyl, and alkynyl groups, forexample. The term “cyclic group” means a closed ring hydrocarbon groupthat is classified as an alicyclic group, aromatic group, orheterocyclic group. The term “alicyclic group” means a cyclichydrocarbon group having properties resembling those of aliphaticgroups. The term “aromatic group” refers to mono- or polycyclic aromatichydrocarbon groups. As used herein, an organic group can be substitutedor unsubstituted.

[0017] The terms “polypeptide” and “peptide” are used interchangeablyherein to refer to a polymer of amino acids. These terms do not connotea specific length of a polymer of amino acids. Thus, for example, theterms oligopeptide, protein, and enzyme are included within thedefinition of polypeptide or peptide, whether produced using recombinanttechniques, chemical or enzymatic synthesis, or naturally occurring.This term also includes polypeptides that have been modified orderivatized, such as by glycosylation, acetylation, phosphorylation, andthe like.

[0018] The following abbreviations are used throughout the application:A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys =Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N =Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe =Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu =Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R= Arg = Arginine S = Ser = Serine H = His = Histidine

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention can be better understood with reference to thefollowing detailed description together with the appended illustrativedrawings in which like elements are numbered the same:

[0020]FIG. 1. Colostrinin inhibits formation of protein-HNE (i.e., 4-HNEprotein) adducts. (A): 4HNE (25 nM); (B): H₂O₂ (100 EM); (C): CLN(10μg/ml) pre-treatment followed by 4HNE (25 nM) exposure; (D): LAH (10μg/ml) pre-treatment followed by 4HNE (25 nM) exposure; (E): HNE-proteinadducts detected by Western blot analysis. Lane 1, 25 nM; lane 2, 12.5nM; lane 3, 6.2 nM of 4HNE alone; lanes 4-6, CLN (10 μg/ml) plus 4HNE,25, 12.5 and 6.2 nM, respectively.

[0021]FIG. 2. Colostrinin inhibits 4HNE-induced oxidative stress. (A):1, control; 2, colostrinin (10 μg/ml); 3, 4HNE (25 nM); 4, 4HNE (25 nM)plus colostrinin (10 μg/ml); 5, lactalbumin hydrolysate (10 μg/ml); 6,lactalbumin hydrolysate (10 μg/ml) plus 4HNE (25 nM). (B): Arepresentative FACS histogram of fluorescence of cells treated with 4HNE(25 nM) and CLN (10 μg/ml) plus 4HNE.

[0022]FIG. 3. Effect of CLN on 4HNE-induced loss of intracellular GSHlevels. Cells were mock-treated or treated with CLN (or LAH) and/or 4HNEfor 30 min, and o-phthalaldehyde-mediated fluorescence was determined asdescribed in Materials and Methods. Open columns: 1, mock-treated; 2,CLN (10 μg/ml)-; 3, LAH (10 μg/ml)-; 4, 4HNE (25 nM)-treated. Filledsolid columns: 5, CLN (10 μg/ml) pre- and 4HNE (25 nM)-treated for 30min; 6, LAH (10 μg/ml) pre- and 4HNE (25 nM)-treated for 30 min.

[0023]FIG. 4. Inhibition of JNK induction by colostrinin. A change inJNK's phosphotyrosine levels was monitored by SDS-PAGE analysis. Equalamounts of protein (50 μg) were fractionated, blotted, and probed withanti-phospho-(Thr-183/Tyr-185)-JNK antibody. Lanes 1 and 2, mock-treatedcells; lane 3, 8-(4-chlorophenylthio)-cAMP, an inhibitor of JNKactivation; lanes 4 and 5, 25 nM 4HNE; lane 6, CLN (10 μg/ml) alone;lane 7, 25 nM 4HNE plus 10 μg/ml CLN; lane 8,25 nM 4HNE plus 1 μg/mlCLN; lane 9,25 nM 4HNE plus 0.1 μg/ml CLN.

[0024]FIG. 5. CLN reduces 4HNE-mediated activation of p53. PC12 cellswere pre-treated with CLN or LAH and exposed to 4HNE. Three hours aftertreatment, cell lysates were analyzed by Western blot analysis. (B) p53;(A) corresponding α-tubulin. 4HNE (25 nM), CLN (10 μg/ml), LAH (10μg/ml).

[0025]FIGS. 6A-6D. (A) Normal morphology of SH—SYSY control cells. Cellsare mostly clumped, non-contact inhibited (right arrow) with a fewelongated cells present. Their refractability indicates they are healthyand growing normally. (B) Cells treated with Beta-amyloid (10 μg/mladded on day 5) that show its toxicity. Note small round granulatedcells with little refractability. (C) Differentiated SH—SY5Y cellsfollowing treatment with CLN (0.1 μg/ml added on day 5 for 30 minutes).Touching cells are flat, contact inhibited (not clumped), left arrow,and more isolated cells are elongated and neuronal in appearance, rightarrow. (D) Cells protected from toxic (apoptotic effect) of Beta-amyloidby treating with CLN (Colostrinin 0.1 μg/ml added on day 5 for 30minutes+Beta-amyloid 10 μg/ml added on day 5). Cells are flat (upperarrow) or elongated (lower arrow) showing typical morphology ofdifferentiated cells (see FIG. 6C). (E) Inhibition of toxicity (apototicactivity) of Beta-amyloid by CLN treatment (Colostrinin 3 μg/ml added onday 5 for 30 minutes+Beta-amyloid 10 μg/ml added on day 5). Noteflattened (bottom arrow) and elongated (upper arrow) cells typical ofSH-SY5Y differentiated cells. (F) Toxic (apoptotic) effect of retinoicacid (20 μM added on day 1) on SH—SYSY cells. The observed toxicityresembles cytopathology induced by viruses. Cytoplasmic bridging causedby shrinking of cells once in contact with each other (upper rightarrows), shrunken granular cells (lower right arrow) and small roundcells (lower left arrows). (G) Inhibition of toxic effect of retinoicacid by treatment of SH—SY5Y with CLN (20 μM retinoic acid added on day1+1 μg/ml Colostrinin added on day S for 30 minutes). Cells are wellorganized showing typical morphology of differentiated SH—SY5Y cells,elongation (lower arrow) and flattening (upper arrow).

[0026]FIG. 7. Analysis of apoptosis by flow cytometry. (A) Induction ofapoptosis by 4HNE (100 nM). UL, upper left; UR, upper right: necroticcells; LL, lower left: viable cells; LR, lower right: apoptotic cells.(B) Absence of apoptosis in mock-treated cells. UL, upper left; UR,upper right: necrotic cells; LL, lower left: viable cells; LR, lowerright: apoptotic cells.

[0027]FIG. 8. Inhibition of 4HNE-induced apoptosis by CLN. PC12 cellswere treated with CLN (1 μg per ml) for 15 min and 4HNE (100 nM) wasadded. Twenty four hours later, cells were harvested and stained withannexin V-PE and 7-AAD. 1, solvent alone; 2, 100 nM 4HNE; 3, TROLOX(vitamin E) 4, col (internal control)+100 nM 4HNE; 5, CLN alone (1 μgper ml); 6, CLN (1 μg per ml)+100 nM 4HNE.

[0028]FIG. 9. Inhibition of UV-B-induced apoptosis by CLN. Parallelcultures of PC12 cells were treated with CLN (1 μg per ml) or col (1 μgper ml) and exposed to LD50 of UV-B. Twenty four hours later, cells wereharvested and stained with annexin V-PE and 7-AAD. 1, Mock-treated; 2,UV-B (LD50); 3, col (internal control); 4, col +UV-B (LD50); 5, CLNalone (1 μg per ml); 6, CLN (1 μg per ml)+UV-B (LD50).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0029] Colostrinin, a complex of proline-rich polypeptides derived fromovine colostrum, induces mitogenic stimulation and a variety ofcytokines in human peripheral blood leukocytes. It also possessesanti-oxidant activity in pheochromocytoma (PC12) cells.

[0030] It has been discovered that colostrinin, at least one constituentpeptide thereof, and/or at least one active analog thereof (e.g., apeptide having an N-terminal sequence equivalent to an N-terminalsequence of at least one of the colostrinin constituent peptides) can beused as modulators of intracellular signaling mechanisms. The signalingmolecules discovered to date that are modulated include 4HNE adductformation, GSH, P53, and JNK.

[0031] More specifically, the present invention provides methods thatinvolve: 1) reduction of the abundance of 4HNE-protein adducts as shownby fluorescent microscopy and Western blot analysis; 2) reduction ofintracellular levels of ROS as shown by a decrease in2′,7′dichlorodihydro-fluorescein-mediated fluorescence; 3) inhibition of4HNE-mediated glutathione depletion as determined fluorimetrically; and4) inhibition of 4HNE-induced activation of c-Jun NH2-terminal kinases.Furthermore, the present invention provides methods that down regulatethe 4HNE-mediated lipid peroxidation and its product-induced signalingthat otherwise may lead to pathological changes at the cellular andorgan level.

[0032] Also, the present invention relates to the use of colostrinin, atleast one constituent (i.e., component) peptide thereof, at least oneactive analog thereof (e.g., peptide having an N-terminal sequenceequivalent to an N-terminal sequence of at least one of the colostrininconstituent peptides), and combinations thereof, in the inhibition ofapoptosis, specifically, the inhibition is related to the apoptotic(cytotoxic) effect of β-amyloid on SH—SY5Y neuronal cells and TNF-alphaor the apoptotic effect of retinoic acid.

[0033] Such compounds (e.g. modulators such as inhibitors) are referredto herein as “active agents.” Significantly, such active agents can beadministered alone or in various combinations to a patient (e.g.,animals including humans) as a medication or dietary (e.g., nutrient)supplement in a dose sufficient to produce the desired effect throughoutthe patient's body, in a specific tissue site, or in a collection oftissues (organs).

[0034] Colostrinin is composed of peptides, the aggregate of which has amolecular weight range between about 5.8 to about 26 kiloDaltons (kDa)determined by polyacrylamide gel electrophoresis. It has a greaterconcentration of proline than any other amino acid. Ovine colostrininhas been found to have a molecular weight of about 18 kDa and includesthree non-covalently linked subunits having a molecular weight of about6 kDa and has about 22 wt-% proline.

[0035] Colostrinin has been found to include a number of peptidesranging from 3 amino acids to 22 amino acids or more. These can beobtained by various known techniques, including isolation andpurification involving eletrophoresis and synthetic techniques. Thespecific method of obtaining colostrinin and SEQ ID NO:31 is describedin International Publication No. WO 98/14473. Using HPLC and EdelmanDegradation, over 30 constituent peptides of colostrinin have beenidentified, which can be classified into several groups: (A) those ofunknown precursor; (B) those having a β-casein homologue precursor; (C)those having a β-casein precursor; and (D) those having an annexinprecursor. These peptides are described in International PatentPublication No. WO 00/75173, published Dec. 14, 2000, and can besynthesized according to well-known synthetic methods. These peptides(i.e., constituent peptides of colostrinin), which can be derived fromcolostrinin or chemically synthesized, include: MQPPPLP; (SEQ ID NO:1)LQTPQPLLQVMMEPQGD; (SEQ ID NO:2) DQPPDVEKPDLQPFQVQS; (SEQ ID NO:3)LFFFLPVVNVLP; (SEQ ID NO:4) DLEMPVLPVEPFPFV; (SEQ ID NO:5) MPQNFYKLPQM;(SEQ ID NO:6) VLEMKFPPPPQETVT; (SEQ ID NO:7) LKPFPKLKVEVFPFP; (SEQ IDNO:8) VVMEV; (SEQ ID NO:9) SEQP; (SEQ ID NO:10) DKE; (SEQ ID NO:11)FPPPK; (SEQ ID NO:12) DSQPPV; (SEQ ID NO:13) DPPPPQS; (SEQ ID NO:14)SEEMP; (SEQ ID NO:15) KYKLQPE; (SEQ ID NO:16) VLPPNVG; (SEQ ID NO:17)VYPFTGPIPN; (SEQ ID NO:18) SLPQNILPL; (SEQ ID NO:19) TQTPVVVPPF; (SEQ IDNO:20) LQPEIMGVPKVKETMVPK; (SEQ ID NO:21) HKEMPFPKYPVEPFTESQ; (SEQ IDNO:22) SLTLTDVEKLHLPLPLVQ; (SEQ ID NO:23) SWMHQPP; (SEQ ID NO:24)QPLPPTVMFP; (SEQ ID NO:25) PQSVLS; (SEQ ID NO:26)LSQPKVLPVPQKAVPQRDMPIQ; (SEQ ID NO:27) AFLLYQE; (SEQ ID NO:28) RGPFPILV;(SEQ ID NO:29) ATFNRYQDDHGEEILKSL; (SEQ ID NO:30) VESYVPLFP; (SEQ IDNO:31) FLLYQEPVLGPVR; (SEQ ID NO:32) LNF; (SEQ ID NO:33) andMHQPPQPLPPTVMFP. (SEQ ID NO:34)

[0036] These can be classified as follows: (A) those of unknownprecursor include SEQ ID NOs:2, 6, 7, 8, 10, 11, 14, and 33; (B) thosehaving a β-casein homologue precursor include SEQ ID NOs: 1, 3, 4, 5, 9,12, 13, 15, 16, 17, and 31; (C) those having a β-casein precursorinclude SEQ ID NOs:18 (casein amino acids 74-83), 19 (casein amino acids84-92), 20 (casein amino acids 93-102), 21 (casein amino acids 103-120),22 (casein amino acids 121-138), 23 (casein amino acids 139-156), 24(casein amino acids 157-163), 25 (casein amino acids 164-173), 26(casein amino acids 174-179), 27 (casein amino acids 180-201), 28(casein amino acids 202-208), 29 (casein amino acids 214-222), 32(casein amino acids 203-214), and 34 (casein amino acids 159-173); and(D) those having an annexin precursor include SEQ ID NO:30 (annexinamino acids 203-220).

[0037] A preferred group of such peptides includes: MQPPPLP; (SEQ IDNO:1) LQTPQPLLQVMMEPQGD; (SEQ ID NO:2) DQPPDVEKPDLQPFQVQS; (SEQ ID NO:3)LFFFLPVVNVLP; (SEQ ID NO:4) DLEMPVLPVEPFPFV; (SEQ ID NO:5) MPQNFYKLPQM;(SEQ ID NO:6) VLEMKFPPPPQETVT; (SEQ ID NO:7) LKPFPKLKVEVFPFP; (SEQ IDNO:8)

[0038] and combinations thereof.

[0039] The polypeptides of SEQ ID NOs:1-34 can be in their free acidform or they can be amidated at the C-terminal carboxylate group. Thepresent invention also includes analogs of the polypeptides of SEQ IDNOs:1-34, which includes polypeptides having structural similarity withSEQ ID NOs:1-34. These peptides can also form a part of a largerpeptide. An “analog” of a polypeptide includes at least a portion of thepolypeptide, wherein the portion contains deletions or additions of oneor more contiguous or noncontiguous amino acids, or containing one ormore amino acid substitutions. An “analog” can thus include additionalamino acids at one or both of the terminii of the polypeptides listedabove. Substitutes for an amino acid in the polypeptides of theinvention are preferably conservative substitutions, which are selectedfrom other members of the class to which the amino acid belongs. Forexample, it is well-known in the art of protein biochemistry that anamino acid belonging to a grouping of amino acids having a particularsize or characteristic (such as charge, hydrophobicity andhydrophilicity) can generally be substituted for another anino acidwithout substantially altering the structure of a polypeptide.

[0040] For the purposes of this invention, conservative amino acidsubstitutions are defined to result from exchange of amino acidsresidues from within one of the following classes of residues: Class I:Ala, Gly, Ser, Thr, and Pro (representing small aliphatic side chainsand hydroxyl group side chains); Class II: Cys, Ser, Thr and Tyr(representing side chains including an —OH or —SH group); Class III:Glu, Asp, Asn and Gln (carboxyl group containing side chains): Class IV:His, Arg and Lys (representing basic side chains); Class V: Ile, Val,Leu, Phe and Met (representing hydrophobic side chains); and Class VI:Phe, Trp, Tyr and His (representing aromatic side chains). The classesalso include related amino acids such as 3Hyp and 4Hyp in Class I;homocysteine in Class II; 2-aminoadipic acid, 2-aminopimelic acid,(γ-carboxyglutamic acid, β-carboxyaspartic acid, and the correspondingamino acid amides in Class III; ornithine, homoarginine, N-methyllysine, dimethyl lysine, trimethyl lysine, 2,3-diaminopropionic acid,2,4-diaminobutyric acid, homoarginine, sarcosine and hydroxylysine inClass IV; substituted phenylalanines, norleucine, norvaline,2-aminooctanoic acid, 2-aminoheptanoic acid, statine and β-valine inClass V; and naphthylalanines, substituted phenylalanines,tetrahydroisoquinoline-3-carboxylic acid, and halogenated tyrosines inClass VI.

[0041] Preferably, the active analogs of colostrinin and its constituentpeptides include polypeptides having a relatively large number ofproline residues. Because proline is not a common amino acid, a “largenumber” preferably means that a polypeptide includes at least about 15%proline (by number), and more preferably at least about 20% proline (bynumber). Most preferably, active analogs include more proline residuesthan any other amino acid.

[0042] As stated above, active analogs of colostrinin and itsconstituent peptides include polypeptides having structural similarity.Structural similarity is generally determined by aligning the residuesof the two amino acid sequences to optimize the number of identicalamino acids along the lengths of their sequences; gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of identical amino acids, although the amino acids in eachsequence must nonetheless remain in their proper order. Preferably, twoamino acid sequences are compared using the Blastp program, version2.0.9, of the BLAST 2 search algorithm, available athttp://www.ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, the defaultvalues for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on. In the comparison oftwo amino acid sequences using the BLAST search algorithm, structuralsimilarity is referred to as “identity.” Preferably, an active analog ofcolostrinin or its constituent peptides has a structural similarity tocolostrinin or one or more of its constituent peptides (preferably, oneof SEQ ID NOs:1-34) of at least about 70% identity, more preferably, atleast about 80% identity, and most preferably, at least about 90%identity.

[0043] Colostrinin or any combination of its peptide components oractive analogs thereof can be derived (preferably, isolated andpurified) naturally such as by extraction from colostrum or can besynthetically constructed using known peptide polymerization techniques.For example, the peptides of the invention may be synthesized by thesolid phase method using standard methods based on eithert-butyloxycarbonyl (BOC) or 9-fluorenylmethoxy-carbonyl (FMOC)protecting groups. This methodology is described by G. B. Fields et al.in Synthetic Peptides: A User's Guide, W. M. Freeman & Company, NewYork, NY, pp. 77-183 (1992). Moreover, gene sequence encoding thecolostrinin peptides or analogs thereof can be constructed by knowntechniques such as expression vectors or plasmids and transfected intosuitable microorganisms that will express the DNA sequences thuspreparing the peptide for later extraction from the medium in which themicroorganism are grown. For example, U.S. Pat. No. 5,595,887 describesmethods of forming a variety of relatively small peptides throughexpression of a recombinant gene construct coding for a fusion proteinwhich includes a binding protein and one or more copies of the desiredtarget peptide. After expression, the fusion protein is isolated andcleaved using chemical and/or enzymatic methods to produce the desiredtarget peptide.

[0044] The peptides used in the methods of the present invention may beemployed in a monovalent state (i.e., free peptide or a single peptidefragment coupled to a carrier molecule). The peptides may also beemployed as conjugates having more than one (same or different) peptidefragment bound to a single carrier molecule. The carrier may be abiological carrier molecule (e.g., a glycosaminoglycan, a proteoglycan,albumin or the like) or a synthetic polymer (e.g., a polyalkyleneglycolor a synthetic chromatography support). Typically, ovalbumin, humanserum albumin, other proteins, polyethylene glycol, or the like areemployed as the carrier. Such modifications may increase the apparentaffinity and/or change the stability of a peptide. The number of peptidefragments associated with or bound to each carrier can vary, but fromabout 4 to 8 peptides per carrier molecule are typically obtained understandard coupling conditions.

[0045] For instance, peptide/carrier molecule conjugates may be preparedby treating a mixture of peptides and carrier molecules with a couplingagent, such as a carbodiimide. The coupling agent may activate acarboxyl group on either the peptide or the carrier molecule so that thecarboxyl group can react with a nucleophile (e.g., an amino or hydroxylgroup) on the other member of the peptide/carrier molecule, resulting inthe covalent linkage of the peptide and the carrier molecule. Forexample, conjugates of a peptide coupled to ovalbumin may be prepared bydissolving equal amounts of lyophilized peptide and ovalbumin in a smallvolume of water. In a second tube,1-ethyl-3-(3-dimethylamino-propyl)-carboiimide hydrochloride (EDC; tentimes the amount of peptide) is dissolved in a small amount of water.The EDC solution was added to the peptide/ovalbumin mixture and allowedto react for a number of hours. The mixture may then dialyzed (e.g.,into phosphate buffered saline) to obtain a purified solution ofpeptide/ovalbumin conjugate. Peptide/carrier molecule conjugatesprepared by this method typically contain about 4 to 5 peptides perovalbumin molecule.

[0046] The present invention also provides a composition that includesone or more active agents (i.e., colostrinin, at least one constituentpeptide thereof, or active analog thereof) of the invention and one ormore carriers, preferably a pharmaceutically acceptable carrier. Themethods of the invention include administering to, or applying to theskin of, a patient, preferably a mammal, and more preferably a human, acomposition of the invention in an amount effective to produce thedesired effect. The active agents of the present invention areformulated for enteral administration (oral, rectal, etc.) or parenteraladministration (injection, internal pump, etc.). The administration canbe via direct injection into tissue, interarterial injection,intervenous injection, or other internal administration procedures, suchas through the use of an implanted pump, or via contacting thecomposition with a mucus membrane in a carrier designed to facilitatetransmission of the composition across the mucus membrane such as asuppository, eye drops, inhaler, or other similar administration methodor via oral administration in the form of a syrup, a liquid, a pill,capsule, gel coated tablet, or other similar oral administration method.The active agents can be incorporated into an adhesive plaster, a patch,a gum, and the like, or it can be encapsulated or incorporated into abio-erodible matrix for controlled release.

[0047] The carriers for internal administration can be any carrierscommonly used to facilitate the internal administration of compositionssuch as plasma, sterile saline solution, IV solutions or the like.Carriers for administration through mucus membranes can be anywell-known in the art. Carriers for administration oral can be anycarrier well-known in the art.

[0048] The formulations may be conveniently presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the active agent intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

[0049] Formulations suitable for parenteral administration convenientlyinclude a sterile aqueous preparation of the active agent, ordispersions of sterile powders of the active agent, which are preferablyisotonic with the blood of the recipient. Isotonic agents that can beincluded in the liquid preparation include sugars, buffers, and sodiumchloride. Solutions of the active agent can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions of the activeagent can be prepared in water, ethanol, a polyol (such as glycerol,propylene glycol, liquid polyethylene glycols, and the like), vegetableoils, glycerol esters, and mixtures thereof. The ultimate dosage form issterile, fluid, and stable under the conditions of manufacture andstorage. The necessary fluidity can be achieved, for example, by usingliposomes, by employing the appropriate particle size in the case ofdispersions, or by using surfactants. Sterilization of a liquidpreparation can be achieved by any convenient method that preserves thebioactivity of the active agent, preferably by filter sterilization.Preferred methods for preparing powders include vacuum drying and freezedrying of the sterile injectible solutions. Subsequent microbialcontamination can be prevented using various antimicrobial agents, forexample, antibacterial, antiviral and antifungal agents includingparabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.Absorption of the active agents over a prolonged period can be achievedby including agents for delaying, for example, aluminum monostearate andgelatin.

[0050] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as tablets,troches, capsules, lozenges, wafers, or cachets, each containing apredetermined amount of the active agent as a powder or granules, asliposomes containing the active agent, or as a solution or suspension inan aqueous liquor or non-aqueous liquid such as a syrup, an elixir, anemulsion, or a draught. The amount of active agent is such that thedosage level will be effective to produce the desired result in thesubject.

[0051] Nasal spray formulations include purified aqueous solutions ofthe active agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes. Formulations for rectal orvaginal administration may be presented as a suppository with a suitablecarrier such as cocoa butter, or hydrogenated fats or hydrogenated fattycarboxylic acids. Ophthalmic formulations are prepared by a similarmethod to the nasal spray, except that the pH and isotonic factors arepreferably adjusted to match that of the eye. Topical formulationsinclude the active agent dissolved or suspended in one or more mediasuch as mineral oil, DMSO, polyhydroxy alcohols, or other bases used fortopical pharmaceutical formulations.

[0052] Useful dosages of the active agents can be determined bycomparing their in vitro activity and the in vivo activity in animalmodels. Methods for extrapolation of effective dosages in mice, andother animals, to humans are known in the art; for example, see U.S.Pat. No. 4,938,949.

[0053] The tablets, troches, pills, capsules, and the like may alsocontain one or more of the following: a binder such as gum tragacanth,acacia, corn starch or gelatin; an excipient such as dicalciumphosphate; a disintegrating agent such as corn starch, potato starch,alginic acid and the like; a lubricant such as magnesium stearate; asweetening agent such as sucrose, fructose, lactose or aspartame; and anatural or artificial flavoring agent. When the unit dosage form is acapsule, it may further contain a liquid carrier, such as a vegetableoil or a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir maycontain one or more of a sweetening agent, a preservative such asmethyl- or propylparaben, an agent to retard crystallization of thesugar, an agent to increase the solubility of any other ingredient, suchas a polyhydric alcohol, for example glycerol or sorbitol, a dye, andflavoring agent. The material used in preparing any unit dosage form issubstantially nontoxic in the amounts employed. The active agent may beincorporated into sustained-release preparations and devices.

EXAMPLES

[0054] The invention will be further described by reference to thefollowing detailed examples. The examples are meant to provideillustration and should not be construed as limiting the scope of thepresent invention.

Examples 1-5 Materials and Methods

[0055] Cell cultures: Pheochromocytoma (PC12) cells were provided by Dr.Regino Perez-Polo (University of Texas Medical Branch, Department ofHuman Biological Chemistry and Genetics) and maintained in EMEMsupplemented with 10% fetal bovine serum, penicillin (100 IU/mI) andstreptomycin (100 micrograms per milliliter (μg/ml)). Exponentiallygrowing populations of PC12 cells were sub-cultured and used for allexperiments.

[0056] Western blot analysis: PC12 cells were plated at 7×10⁶ cells/T75flask. After exposure to 4HNE, colostrinin or their combination, cellswere collected and lysed in 50 millimolar (mM) Tris, 5 mM EDTA, 150 mMNaCl, 0.5% NP-40, 10% glycerol and protease inhibitor cocktail(supplemented with 1 mM Na₃VO₄, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride). Lysates were centrifuged at 14,000g for 10 minutes (min) (4°C.) and 40 μg of protein was fractionated on a 10% SDS-polyaciylamidegel and transferred to protein-optimized membranes (Amersham, Inc.). p53was detected using specific antibody (DO1; Santa Cruz Biotechnology,Inc.) at a dilution of 1:300. Adducts were detected using an antibody toHNE-protein adducts (Phanningen, Inc.) at a dilution of 1:500. Theanti-phospho-JNK antibody (New England Biolabs, Inc., Beverly, Mass.)was raised against a synthetic phosphopeptide (SFMMT*PY*VVTRYYR)corresponding to residues 179-193 of JNK. For visualization of primaryantibody binding, all blots were incubated with horseradishperoxidase-conjugated secondary antibody (Amersham, Inc.) at a dilutionof 1:2000, followed by chemiluminescence detection (Amersham, Inc.) andautoradiography.

[0057] Immunocytochemistry: PC12 cells grown on cover-slips were fixedovernight in PBS containing 2% paraformaldehyde at 4° C. Cells werepermeabilized by 0.3% Triton X-100, washed in PBS then incubated withprimary antibody in PBS containing 0.05% Tween 20 (PBS-7). After washing3 times in PBS-T, FITC-labeled anti-rabbit IgG (Santa Cruz BiotechnologyInc.) was added. Cells were washed (5 times, for 10 min) with PBS-T andmounted on microscope slides in anti-fade solution (Dako, Inc.). Imagesof cellular immunofluorescence were acquired using a NIKON Eclipse TE300scanning microscope.

[0058] Measurement of glutathione (GSH): In brief, PC12 cells were mock-or pre-treated with CLN or lactalbumin hydrolysate (LAH), both in 10μg/ml concentration and then exposed to 4HNE (25 nM). PBS-washed (twice)cells were then extracted with 25% (w/v) metaphosphoric acid solutioncontaining 5 mM EDTA. After ultracentrifugation (105,000g for 30 min),100 μd of 100 mM phosphate solution (pH 8.0) containing 5 mM EDTA and 10μd of o-phthalaldehyde OPA(OPD; Molecular Probes, Inc.) was added to thesupernatant, and the fluorescence intensity at 420 nm determined withexcitation set at 350 nm (A. P. Senft et al., Anal Biochem., 280:80-86(2000)).

[0059] Flow cytometry: Relative changes in ROS levels were determined asdescribed previously (I. Boldogh et al., Psychogeriatr Ann, 4:57-65(2001)). Briefly, PC12 cells at 70% confluence were trypsinized andwashed with EMEM containing 10% FBS. Cells were re-suspended in EMEM(plus 5% FBS) and loaded with 2′,7′dichlorodihydro-fluorescein diacetate(H₂DCF-DA; Molecular Probes Inc.) (5 mM final concentration) for 15 min,at 37° C. then washed in growth medium. Following centrifugation, thecell pellets were re-suspended in EMEM containing 10 mM HEPES (pH: 7.4).DCF-mediated fluorescence of treated and mock-treated cells wasdetermined by flow cytometry (Becton Dickinson FACS Scan) using 488 nmand 525 nm excitation and emission settings, respectively. Each datapoint represents the mean fluorescence for 12,000 cells.

[0060] Reagents: Colostrinin (CLN) was purified from ovine colostrum,collected during the first milking (6-12 hours (hr) after lambing),according to the method developed by Janusz et al. (M. Janusz et al.,FEBS Lett., 49:276-279 (1974)). A high content of proline (>23%) andlack of detectable alanine, arginine, histidine, tryptophan, methionine,and cysteine were confirmed by amino acid analysis of CLN. A peptidecontrol was prepared by trypsin (Sigma-Aldrich) digestion of purifiedlactalbumin from bovine milk (Sigma). The trypsin was then inhibited bytreatment with inhibitor (Invitrogen). SDS-PAGE confirmed digestion oflactalbunin into peptides, and the hydrolysate was referred to as LAH.

[0061] Statistical analysis: The experiments were repeated at leastthree times and statistically analyzed for significant differences usingANOVA procedures and Student's t-tests. Data are expressed as means±S.E.

Examples 1-5 Results Example 1 Colostrinin Reduces 4HNE-Protein AdductFormation in PC 12 Cells.

[0062] Fluorescent microscopy and Western blot analysis was undertakento investigate the extent of 4HNE protein-adduct formation in culturesof PC12 cells in the presence of CLN. Cells were pretreated with CLN orLAH in the presence or absence of 4HNE and then analyzed for theformation of 4HNE-protein adducts. The results in FIG. 1A and 1B showthat addition of 4HNE (25 nanomolar (nM)) or H₂O₂ (100 micromolar (μM))resulted in a bright fluorescence, localized to the cytoplasmic regionof PC12 cells due to binding of antibody to 4HNE-protein adducts. Whencells were pre-treated with CLN (10 microgram per milliliter (μg/ml))for 15 minutes (min) and exposed to 4HNE (25 nM) for 15 min(concentrations of CLN and time required for effect were determined inpreliminary studies), the results indicated that CLN reducedfluorescence intensity (FIG. 1C) to background level (data not shown).In the controls, pre-treatment of cells with an N-acetyl-L-ysteine (10mM) and trolox (1 mM; a water-soluble -tocopherol) combinationsignificantly reduced 4HNE-mediated intracellular fluorescence.

[0063] To determine whether the inhibitory effect of CLN was specific,CLN was substituted with digested lactalbumin hydrolysate (LAH,Materials and Methods), which contains a variety of peptides as doesCLN. Results in FIG. ID show bright fluorescence in cells treated withLAH (10 μg/nml) plus 4HNE (25 nM), which is similar to that seen with4HNE alone (FIG. 1A). These data indicate that CLN inhibits adductformation, and the effect is specific and could be the result of a notyet-determined interaction between its constituent peptides and cellularcomponent(s).

[0064] To confirm the results generated by immunochemistry, Western blotanalysis was used to investigate changes in 4HNE-protein adduct levelsin cells treated with 4HNE alone or CLN plus 4HNE. FIG. 1E (lanes 1, 2,and 3) shows that 4HNE alone induced a significant increase in levels of4HNE-protein adducts, with molecular weights ranging from 200 kD to 15kD. CLN (10 μg/ml) abolished adduct formation, as shown in FIG. 1E lanes4 to 6. Overall these data indicate that CLN can block the formation of4HNE-adducts. From these results, it is believed that the inhibition of4HNE-protein adduct formation by CLN is multi-factorial and may involvemechanisms such as direct scavenging (binding) of 4HNE via cysteine,lysine, or histidine residues in CLN, or by inhibition of 4HNE's entryonto cells.

[0065] To determine whether CLN can protect mitochondria and abolish theoxidative stress induced by 4HNE, PC 12 cells were treated with CLN(with LAH as control) and/or 4HNE and the changes in ROS levels weremonitored by the redox-sensitive 2′,7′-dichlorofluorescein diacetate(H₂DCF-DA) probe (I. Boldogh et al., Psychogeriatr Ann., 4:57-65(2001);LeBel, Chem. Res. Toxicol., 5:227-231 (1992)). Mock- as well as CLN (orLAH) pre-treated cells were loaded with H₂DCF-DA then exposed to 4HNEfor 15 min. Changes in fluorescence intensities mediated by the oxidizedprobe, DCF, were determined by flow cytometry. Prior to data collection,propidium iodide was added to the samples for sorting out nonviablecells.

Example 2 Colostrinin Affects the Oxidative Metabolism in PC12 Cells

[0066] A representative histogram showing the effects of the treatmentson ROS levels is shown in FIG. 2B. As summarized in FIG. 2A, 4HNE (25nM) induced a 4- to 5-fold increase in DCF-mediated fluorescence, whileCLN alone or LAH showed no significant effect. Remarkably, CLN abolished(while LAH had no significant effect on) H₂DCF oxidation in 4HNE-treatedPC 12 cells. Because constituent peptides in LAH did not alter4HNE-induced H₂DCF oxidation, it can be concluded that the effect of CLNis specific, and may protect cells from ROS damage via itsquantitatively unique and specific peptide composition.

Example 3 Effect of CLN on 4HNE -Induced Loss of Intracellular GSHLevels

[0067] To investigate whether the anti-oxidant effect of CLN was due toprotection of intracellular GSH levels, PC12 cells were pre-treated(with CLN or LAH) and exposed to 4HNE, as described above, and changesin GSH levels were determined fluorimetrically. The results summarizedin FIG. 3 show that treatment with 4HNE alone for 30 min (timedetermined in preliminary studies) resulted in a significant reductionof intracellular GSH levels as shown by a change in OPA-GSH'sfluorescence. OPA (o-phthalaldehyde or phthalic dicarboxaldehyde) ishighly fluorescent when it is conjugated to GSH (A.P. Senft et al., AnalBiochem., 280:80-86 (2000)). Pre-treatment of cells with CLN (10 μg/ml),however, significantly inhibited this change in OPA fluorescence (lossof GSH) while LAH had an insignificant effect.

[0068] To determine whether the loss of intracellular GSH was due toit's extrusion from the cells or oxidation, the level of reduced GSH inthe extra-cellular fluid was evaluated. Relative to CLN- or mock-treatedcells, 4HNE caused a significant increase in OPA-GSH-mediatedfluorescence when it was added to extracellular fluid (data not shown).LAH alone or LAH in 4HNE-exposed cells did not affect GSH extrusion(OPA-GSH fluorescence). OPA did not show fluorescence when it was mixedwith CLN, LAH or 4HNE alone. These results indicate that CLN mediatesits effect on GSH metabolism at the cell membrane level.

Example 4 4HNE-Induced Activation of JNK is Suppressed by CLN

[0069] The effect of CLN on 4HNE-induced activation of JNK in PC12 cellswas investigated. The activation of JNK was monitored by Western blotanalysis using a highly specific anti-phospho- (Thr-183/Tyr-185) JNKantibody (Materials and Methods).

[0070] The data summarized in FIG. 4 show that 4HNE alone is a potentinducer of JNK phosphorylation (FIG. 4, lanes 4 and 5). Pretreatment ofPC 12 cells with CLN (1 and 10 μg/ml) or with an inhibitor of JNKactivation [8-(4-chlorophenylthio)-cAMP] prevented 4HNE-induced JNKphosphorylation; 4HNE-mediated phosphorylation was reduced by 10 and 1.0μg/ml CLN (FIG. 4, lanes 7 and 8) to control levels (FIG. 4 lanes 1 and2). CLN at 0.1 μg/ml concentration did not significantly effect4HNE-mediated JNK phosphorylation (lane 9). The maximum level ofphosphorylation of JNK in 4HNE-treated cells occurred between 15 and 30min post-treatment as determined in preliminary studies (data notshown). These data indicate that CLN may modulate oxidative metabolism(GSH levels, 4HNE-protein adduct formation) of cells potentially throughJNK, a kinase that is central to the cellular stress responses.

Example 5 Colostrinin Inhibits 4HNE-Induced Activation of p53

[0071] Whether CLN could modulate p53 levels after 4HNE exposure wasalso investigated. In FIG. 5, Western blot analysis shows that CLNreduces activation of p53 induced by 4HNE when compared to cells treatedwith 4HNE alone. On the other hand, pre-treatment with the sameconcentration (10 μg/ml) of LAH had no affect on 4HNE-mediated p53induction. These data suggest that CLN, via its antioxidant activity,can effect activation of p53, a key regulator of cell proliferation,differentiation and apoptosis (G. Evan et al., Science, 281:1317-1322(1998)) and may explain multiple biological effects (antioxidant,differentiation) of CLN.

Examples 1-5 Discussion

[0072] It has been shown that CLN, a milk-derived peptide complex canmodulate both cytokine production and cellular redox status. To studyCLN's antioxidant effects, 4HNE was used for treatment of PC12 cells.4HNE is a 3-unsaturated aldehyde generated endogenously during lipidperoxidation, specifically from the oxidative degradation of arachidonicand linoleic acids (H. Esterbauer et al., Free Radic. Biol. Med,11:81-128 (1991)). Further, 4HNE is involved in both normal andpathophysiological events in cells and tissues that result in variouschronic diseases. While micromolar concentrations of 4HNE is cytotoxic,at the nanomolar level it can be involved in activation of the signaltransduction pathways. For example, it has been shown that depending onconcentration, 4HNE can affect proliferation and induce differentiationor apoptosis in cells.

[0073] In the current studies, a concentration of 25 nM 4HNE was used.This did not show toxic effects or induce apoptosis but considerablyincreased the levels of 4HNE-protein adducts (FIG. 1A) in PC12 cells.Remarkably, it was found that CLN abolished 4HNE-protein adductformation, while LAH (as control) at the same concentration had noeffect indicating CLN's specificity. Due to the presence of a highlyelectrophilic carbon, 4HNE is a potent alkylating agent able to reactwith histidine, lysine, serine, cystein, and tyrosine side chains inproteins, and thus modify their functions. Although it was hypothesizedthat peptides of CLN, via its component amino acid residues, werereacting with 4HNE and chemically reducing its concentration, no directinteraction between CLN's peptide(s) and 4HNE were observed.

[0074] 4HNE is known to modulate the activities of ATPases,phospholipase C, adenylate cyclase, GTP-binding proteins, and proteinkinase C. Furthermore, 4HNE can react with the nucleophilic sites inDNA, mitochondrial proteins and a variety of other nucleophiles,including GSH, resulting in cellular stress responses and oxidativestress. In the present studies, it was demonstrated that that CLN wasable to prevent a decrease in 4HNE-induced GSH levels. It is proposedthat 4HNE-induced reduction in GSH levels may be due toglutathione-S-transferase (GST)mediated conjugation of 4-HNE to GSH, orthat GSH may be utilized in detoxification reactions of ROS.

[0075] Taking into consideration that only 25 nM of 4HNE was used, whileintracellular concentrations of GSH are in the 0.5 to 10 mM range,reduction of GSH levels by GST or utilization by glutathione peroxidasesmay not explain the more than 50% loss of GSH. Therefore, the GSH levelsin the extracellular fluid was evaluated. The large reduction in GSHlevels (FIG. 3) may be due to extrusion of GSH from cells after 4HNEexposure. Indeed, it has been discovered that an increase in GSH in theextracellular milieu is in response to 4HNE treatment. Most remarkably,CLN was able to prevent this effect of 4HNE.

[0076] Although some ROS production in 4HNE-treated cells has been shownto be due to mitochondrial damage, it is believed that the 3- to 4-foldincrease in ROS levels were due to GSH extrusion, which resulted in aperturbance of cellular anti-oxidant defenses. Most importantly, CLN,but not LAH, inhibited oxidation of H₂DCF strongly suggesting that CLNis involved in the activation of cellular antioxidant defenses orpossesses effective anti-oxidant activity via regulating cellular GSHlevels.

[0077] 4HNE exposures have been reported to be linked with c-JunNH2-terminal kinases activation and c-Jun phosphorylation. Three groupsof mitogen-activated protein (MAP) kinases have been identified inmammals: the extracellular signal-regulated kinase, the p38 MAP kinase,and JNKs (also referred to SAPKs). JNKs are activated by a wide varietyof stimuli, including ROS, DNA-damaging agents and inhibitors of proteinsynthesis, and heat or osmotic shock. These stimuli appear to operatethrough small G proteins of the Ras and epidermal growth factor (EGF)family receptors and sequential activation of various protein kinases.Targets of the JNK signal transduction pathway include the transcriptionfactors ATF2 and c-Jun. c-Jun binds to the N-terminal region of ATF2 andc-Jun and phosphorylates two sites within the activation domain. Thesefactors are members of the basic leucine zipper group that binds ashomo- and heterodimeric complexes to AP-1 and AP-1-like sites in thepromoters of many genes and result in increased transcriptionalactivity.

[0078] The present studies show that treatment of PC12 cells with 4HNEcauses JNK activation within 15 to 30 min. However, in CLN pre-treatedcells JNK activation was not only delayed or reduced, it was abolished.CLN was also as potent as 8-(4-chlorophenylthio)-cAMP a specificinhibitor of JNK activation. AP-l phosphorylation, which is a laterevent in the JNK signaling pathway is presently under investigation.These findings are consistent with the idea that CLN has the ability toprotect cells from oxidative stress and other consequences of 4HNEexposure, including JNK activation, and its down-stream consequences.

[0079] The Western blot analysis shown in FIG. 5, clearly demonstratedthat p53 is normally present in a latent form and that 4HNE induced itsactivation. It has been shown that p53 lies at the center of a networkof complex redox interactions. In this network, p53 can control thetimely production of ROS, but this activity is itself under the controlof changes in cellular redox status. Thus, p53 activation in4HNE-treated PC12 cells can occur in multiple ways: it may be due to4HNE-induced DNA damage, ROS, and/or activation of cell cycle regulatorykinases. Regardless of the mechanism of p53 activation, CLN showed apotent inhibitory effect.

[0080] CLN induced differentiation in SH—SY5Y cells in a dose dependantmanner (Table 1A). The ability of CLN to induce differentiation in thesecells is shown in FIG. 6A, the control compared to FIG. 6C, a culturetreated with 0.1 μg/ml CLN (see figure legends).

[0081] It has been shown that colostrinin can inhibit the toxicity ofBeta-amyloid in neural derived SH—SY5Y cells. Essentially completeinhibition of the toxicity occurred at the 0.01 μg/ml level (Table 1B).FIGS. 6D and 6E shows the protective effect of 3.0 and 0.1 μg/ml of CLNon B-amyloid induced toxicity as shown in FIG. 6B. Since this toxicityis the result of the apoptotic activity of Beta-amyloid (β-amyloid), thedata indicate that colostrinin is a potent inhibitor of apoptosis inneural-derived cells. This potent activity indicated that even lowerconcentrations of colostrinin would have to be tested to determine thepotency and anti-apoptotic dose response effect of colostrinin in thissystem. However, a dose dependant development of differentiation didoccur in the presence of Beta-amyloid in the colostrinin treated cells(FIGS. 6D and 6E).

[0082] The results indicate that not only did colostrinin inhibit thetoxicity of Beta-amyloid, but it also was able to induce differentiationof the SH-SYSY in a dose dependant manner in Beta-amyloid treated cells.This finding indicates that the development of differentiation inBeta-amyloid treated cells could be used as a biological assay forcolostrinin and one of its important functions.

[0083] To determine whether this was reproducible and to determine thepotency of CLN to inhibit retinoic acid toxicity, two concentration ofretinoic acid were used to treat cells on day one of the experiment, 20μM and 40 μM. *Control wells were mock-treated for the duration of theexperiment. Retinoic Acid was left on the plate for the duration of theexperiment or until washed off. Colostrinin was added to the plate atthe indicated doses. When added on Day 1, it was present during theentire experiment. When added on Day 5, it was incubated on the platefor 30 minutes at 37° C. and then removed. The wells were washed twicewith PBS before adding β-Amyloid. Cultures were then observed under themicroscope and graded.

[0084] The data reported herein shows the ability of CLN to block theactivity of cells treated with 20 μM retinoic acid (the toxicitydeveloped too rapidly in cells treated with 40 μM). Table IC indicatesthat 1.0. μg/ml of CLN almost completely blocked the cytotoxicity ofretinoic acid. The retinoic acid induced differentiation in these cellsby day two, but the cells started showing signs of toxicity by day 6.CLN, 1.0 μg/ml, added on day one or for 30 minutes on day five of theexperiment completely blocked the toxicity, and similar to the findingwith Beta-amyloid, also induced the cells to proliferate. FIGS. 6F and6G further document the finding. FIG. 6F shows the toxicity induced byretinoic acid compared to FIG. 6G, which clearly shows differentiatedcells (see legends).

[0085] Colostrinin inhibited the eventual development of cytotoxicity byRetinoic Acid when added at the same time or 5 days later. Colostrininadded at Day 1 also inhibited the development of toxicity by β-Amyloidadded on Day 5 and was dose dependent (data not shown).

[0086] The ability of CLN to inhibit apoptotic effects of twosubstances, Beta-amyloid and retinoic acid, indicates it may havepotential biological use in many areas where apoptosis plays a role,e.g., virus infections, chronic diseases and attempts to get stem cellsto grow and differentiate, among many others. TABLE 1 Differentiating,Anti-Apoptotic and Protective Activity of Colostrinin Against β-Amyloidand Retinoic Acid in Neuronal Derived SH-SY5Y Cells DOSE COLOSTRININTREATMENT (MG/ML) TOXICITY DIFFERENTIATION A. Differentiating Activity3.0 (Day 5)^(a) − (Day 8)^(b) ++++ (Day 8)^(b) 1.0 (Day 5)^(a) − (Day8)^(b) ++++ (Day 8)^(b) 0.1 (Day 5)^(a) − (Day 8)^(b) +++ (Day 8)^(b)0.01 (Day 5)^(a) +/− (Day 8)^(a) ++ (Day 8)^(b) B. Anti-apoptoticActivity β-Amyloid 10 μg/ml (Day 5)^(a) +++ (Day 8)^(b) − (Day 8)^(b)β-Amyloid 10 μg/ml (Day 5)^(a) 3.0 (Day 5)^(a) − (Day 8)^(b) +++ (Day8)^(b) ” 1.0 (Day 5)^(a) +/− (Day 8)^(b) +++ (Day 8)^(b) ” 0.1 (Day5)^(a) +/− (Day 8)^(b) ++ (Day 8)^(b) ” 0.01 (Day 5)^(a) + (Day 8)^(a) +(Day 8)^(b) C. Protection Against Retinoic Acid Activity Retinoic Acid20 μM (Day 1)^(a) − (Day 2)^(b) ++++ (Day 2)^(b) Retinoic Acid 20 μM(Day 1)^(a) +++ (Day 8)^(b) + (Day 8)^(b) Retinoic Acid 20 μM (Day1)^(a) 1.0 (Day 5)^(a) − (Day 8)^(b) ++++ (Day 8)^(b) ” 1.0 (Day1)^(a) + (Day 8)^(b) ++++ (Day 8)^(b) D. Control Mock Treated (Day1)^(a) − (Day 5 & 8)^(b) − (Day 5 & 8)^(b)

Example 7 Inhibition of 4HNE-Induced Apoptosis by CLN

[0087] Apoptosis is a specific mode of cell death recognized by acharacteristic pattern of morphological, biochemical, and molecularchanges. Currently the hallmark of apoptosis in vitro is DNAfragmentation and changes in plasma membrane reorganization that allowsfor the surface expression of phosphatidyl-D-serine and result inincreased membrane permeability. The protection of cells against 4HNE byCLN using increased permeability and cell membrane expression ofphosphatidyl-D-serine was investigated.

[0088] Cells were simultaneously labeled with fluorochrome-conjugatedannexin V-PE (detecting PS asymmetry in the plasma membrane, an earlymarker of apoptosis), 7-aminoactinomycin D (7-AAD) (detecting increasedmembrane permeability associated with both apoptosis and necrosis). Adual laser flow cytometer (either a Becton-Dickinson FACScan) was usedfor the simultaneous detection of the PE-conjugated annexin V (which isexcited at 632 nm and emits at 660 nm), 7-AAD (excited at 488 nm andemitting at 670 nm). Fluorochrome compatibility was excellent, althoughcareful intralaser compensation is required for simultaneous use of PEand 7-AAD.

[0089] The analysis of apoptosis by flow cytometry is shown in FIG. 7.The results of the inhibition of 4HNE-induced apoptosis by CLN are shownin FIG. 8.

Example 8 Inhibition of UV-Irradiation-Induced Apoptosis by CLN

[0090] Chronic repeated UV exposures are the primary cause of benign andmalignant skin tumors, including malignant melanoma. In experimentalanimal models, among types of solar radiation, ultraviolet B (290-320nm) radiation is highly mutagenic and carcinogenic compare toultraviolet A (320400 nm) radiation. Based on current understanding ofDNA damage caused by direct UV radiation and by indirect stress viareactive oxygen species and DNA repair mechanisms are responsible for UVirradiation-induced skin tumor development in human cells.

[0091] UVB exposure leads to a time-dependent increase in the productionof intracellular peroxide and superoxide anions and may inducecarcinogenic mutations and apoptosis. Besides being a major cause ofoxidative stress in the cells, UVB-irradiation induces apoptosis by alarge number of unrelated pathways such as enhanced Fas transcriptionand/or mRNA stability, induction of transcriptional factors viz c-fos,c-jun, SAP-1 and nuclear factor kB gene expression. A possibleprevention of UV- induced skin cancer by feeding or topical use ofantioxidants, such as polyphenols, and vitamins are observed.

[0092] The effect of CLN on ultraviolet B (UVB)-induced apoptosis andDNA damage in cultured PC12 cells has been determined. The apoptosis wasdetermined by flow cytometry. The comet assay was employed to detect DNAdamage in individual cell. The results shown in FIG. 9 indicate aninhibitory effect of CLN on UVB-induced apoptosis. CLN-treated cellsalso showed a significantly reduced DNA damage. Semi-confluent cellswith >98% viability (tested with trypan blue dye exclusion) were used inall experiments. PC12 cells were exposed to UV-B irradiation. Lethaldose 50 (LD50) was determined in preliminary studies. Cells wereirradiated by a dose result in 50% cell death.

[0093] Conclusion

[0094] Taken together, these results show that CLN can be involved inregulation of the cellular redox status, GSH metabolism, and modulationof ROS-induced signaling-mediated down-stream events (e.g., JNK, p53).These results further suggest a potential mechanism(s) by which CLNcould modulate a network, resulting in cytokine, chemokine production,cell differentiation and may explain it's beneficial effects onpathogenic processes involved in AD and other chronic neuro-degenerativediseases.

[0095] Although the invention has been disclosed with reference to itspreferred embodiments, from reading this description those of skill inthe art may appreciate changes and modification that may be made whichdo not depart from the scope and spirit of the invention as describedabove and claimed hereafter. All references, patents, and patentapplications cited herein are incorporated herein by reference in theirentirety as if individually incorporated.

Sequence Listing Free Text

[0096] The following are all synthetic peptide sequences. SEQ ID NO:1MQPPPLP SEQ ID NO:2 LQTPQPLLQVMMEPQGD SEQ ID NO:3 DQPPDVEKPDLQPFQVQS SEQID NO:4 LFFFLPVVNVLP SEQ ID NO:5 DLEMPVLPVEPFPFV SEQ ID NO:6 MPQNFYKLPQMSEQ ID NO:7 VLEMKFPPPPQETVT SEQ ID NO:8 LKPFPKLKVEVFPFP SEQ ID NO:9VVMEV SEQ ID NO:10 SEQP SEQ ID NO:11 DKE SEQ ID NO:12 FPPPK SEQ ID NO:13DSQPPV SEQ ID NO:14 DPPPPQS SEQ ID NO:15 SEEMP SEQ ID NO:16 KYKLQPE SEQID NO:17 VLPPNVG SEQ ID NO:18 VYPFTGPIPN SEQ ID NO:19 SLPQNILPL SEQ IDNO:20 TQTPVVVPPF SEQ ID NO:21 LQPEIMGVPKVKETMVPK SEQ ID NO:22HKEMPFPKYPVEPFTESQ SEQ ID NO:23 SLTLTDVEKLHLPLPLVQ SEQ ID NO:24 SWMHQPPSEQ ID NO:25 QPLPPTVMFP SEQ ID NO:26 PQSVLS SEQ ID NO:27LSQPKVLPVPQKAVPQRDMPIQ SEQ ID NO:28 AFLLYQE SEQ ID NO:29 RGPFPILV SEQ IDNO:30 ATFNRYQDDHGEEILKSL SEQ ID NO:31 VESYVPLFP SEQ ID NO:32FLLYQEPVLGPVR SEQ ID NO:33 LNF SEQ ID NO:34 MHQPPQPLPPTVMFP

[0097]

1 34 1 7 PRT artificial Synthetic Peptides 1 Met Gln Pro Pro Pro Leu Pro1 5 2 17 PRT artificial Synthetic Peptides 2 Leu Gln Thr Pro Gln Pro LeuLeu Gln Val Met Met Glu Pro Gln Gly 1 5 10 15 Asp 3 18 PRT artificialSynthetic Peptides 3 Asp Gln Pro Pro Asp Val Glu Lys Pro Asp Leu Gln ProPhe Gln Val 1 5 10 15 Gln Ser 4 12 PRT artificial Synthetic Peptides 4Leu Phe Phe Phe Leu Pro Val Val Asn Val Leu Pro 1 5 10 5 15 PRTartificial Synthetic Peptides 5 Asp Leu Glu Met Pro Val Leu Pro Val GluPro Phe Pro Phe Val 1 5 10 15 6 11 PRT artificial Synthetic Peptides 6Met Pro Gln Asn Phe Tyr Lys Leu Pro Gln Met 1 5 10 7 15 PRT artificialSynthetic Peptides 7 Val Leu Glu Met Lys Phe Pro Pro Pro Pro Gln Glu ThrVal Thr 1 5 10 15 8 15 PRT artificial Synthetic Peptides 8 Leu Lys ProPhe Pro Lys Leu Lys Val Glu Val Phe Pro Phe Pro 1 5 10 15 9 5 PRTartificial Synthetic Peptides 9 Val Val Met Glu Val 1 5 10 4 PRTartificial Synthetic Peptides 10 Ser Glu Gln Pro 1 11 3 PRT artificialSynthetic Peptides 11 Asp Lys Glu 1 12 5 PRT artificial SyntheticPeptides 12 Phe Pro Pro Pro Lys 1 5 13 6 PRT artificial SyntheticPeptides 13 Asp Ser Gln Pro Pro Val 1 5 14 7 PRT artificial SyntheticPeptides 14 Asp Pro Pro Pro Pro Gln Ser 1 5 15 5 PRT artificialSynthetic Peptides 15 Ser Glu Glu Met Pro 1 5 16 7 PRT artificialSynthetic Peptides 16 Lys Tyr Lys Leu Gln Pro Glu 1 5 17 7 PRTartificial Synthetic Peptides 17 Val Leu Pro Pro Asn Val Gly 1 5 18 10PRT artificial Synthetic Peptides 18 Val Tyr Pro Phe Thr Gly Pro Ile ProAsn 1 5 10 19 9 PRT artificial Synthetic Peptides 19 Ser Leu Pro Gln AsnIle Leu Pro Leu 1 5 20 10 PRT artificial Synthetic Peptides 20 Thr GlnThr Pro Val Val Val Pro Pro Phe 1 5 10 21 18 PRT artificial SyntheticPeptides 21 Leu Gln Pro Glu Ile Met Gly Val Pro Lys Val Lys Glu Thr MetVal 1 5 10 15 Pro Lys 22 18 PRT artificial Synthetic Peptides 22 His LysGlu Met Pro Phe Pro Lys Tyr Pro Val Glu Pro Phe Thr Glu 1 5 10 15 SerGln 23 18 PRT artificial Synthetic Peptides 23 Ser Leu Thr Leu Thr AspVal Glu Lys Leu His Leu Pro Leu Pro Leu 1 5 10 15 Val Gln 24 7 PRTartificial Synthetic Peptides 24 Ser Trp Met His Gln Pro Pro 1 5 25 10PRT artificial Synthetic Peptides 25 Gln Pro Leu Pro Pro Thr Val Met PhePro 1 5 10 26 6 PRT artificial Synthetic Peptides 26 Pro Gln Ser Val LeuSer 1 5 27 22 PRT artificial Synthetic Peptides 27 Leu Ser Gln Pro LysVal Leu Pro Val Pro Gln Lys Ala Val Pro Gln 1 5 10 15 Arg Asp Met ProIle Gln 20 28 7 PRT artificial Synthetic Peptides 28 Ala Phe Leu Leu TyrGln Glu 1 5 29 8 PRT artificial Synthetic Peptides 29 Arg Gly Pro PhePro Ile Leu Val 1 5 30 18 PRT artificial Synthetic Peptides 30 Ala ThrPhe Asn Arg Tyr Gln Asp Asp His Gly Glu Glu Ile Leu Lys 1 5 10 15 SerLeu 31 9 PRT artificial Synthetic Peptides 31 Val Glu Ser Tyr Val ProLeu Phe Pro 1 5 32 13 PRT artificial Synthetic Peptides 32 Phe Leu LeuTyr Gln Glu Pro Val Leu Gly Pro Val Arg 1 5 10 33 3 PRT artificialSynthetic Peptides 33 Leu Asn Phe 1 34 15 PRT artificial SyntheticPeptides 34 Met His Gln Pro Pro Gln Pro Leu Pro Pro Thr Val Met Phe Pro1 5 10 15

What is claimed is:
 1. A method of modulating an intracellular signalingmolecule in a cell, the method comprising contacting the cell with amodulator selected from the group of colostrinin, a constituent peptidethereof, an active analog thereof, and combinations thereof, underconditions effective to accomplish at least one of the following: reduce4HNE-protein adduct formation; inhibit 4HNE-mediated glutathionedepletion; inhibit 4HNE-induced activation of p53 protein; or inhibit4HNE-induced activation of c-Jun NH2-terminal kinases.
 2. The method ofclaim 1 wherein the cell is present in a cell culture, a tissue, anorgan, or an organism.
 3. The method of claim 1 wherein the cell is amammalian cell.
 4. The method of claim 3 wherein the cell is a humancell.
 5. The method of claim I wherein the modulator is a constituentpeptide of colostrinin.
 6. The method of claim 5 wherein the modulatoris selected from the group of MQPPPLP, (SEQ ID NO:1) LQTPQPLLQVMMEPQGD,(SEQ ID NO:2) DQPPDVEKPDLQPFQVQS, (SEQ ID NO:3) LFFFLPVVNVLP, (SEQ IDNO:4) DLEMPVLPVEPFPFV, (SEQ ID NO:5) MPQNFYKLPQM, (SEQ ID NO:6)VLEMKFPPPPQETVT, (SEQ ID NO:7) LKPFPKLKVEVFPFP, (SEQ ID NO:8) VVMEV,(SEQ ID NO:9) SEQP, (SEQ ID NO:10) DKE, (SEQ ID NO:11) FPPPK, (SEQ IDNO:12) DSQPPV, (SEQ ID NO:13) DPPPPQS, (SEQ ID NO:14) SEEMP, (SEQ IDNO:15) KYKLQPE, (SEQ ID NO:16) VLPPNVG, (SEQ ID NO:17) VYPFTGPIPN, (SEQID NO:18) SLPQNILPL, (SEQ ID NO:19) TQTPVVVPPF, (SEQ ID NO:20)LQPEIMGVPKVKETMVPK, (SEQ ID NO:21) HKEMPFPKYPVEPFTESQ, (SEQ ID NO:22)SLTLTDVEKLHLPLPLVQ, (SEQ ID NO:23) SWMHQPP, (SEQ ID NO:24) QPLPPTVMFP,(SEQ ID NO:25) PQSVLS, (SEQ ID NO:26) LSQPKVLPVPQKAVPQRDMPIQ, (SEQ IDNO:27) AFLLYQE, (SEQ ID NO:28) RGPFPILV, (SEQ ID NO:29)ATFNRYQDDHGEEILKSL, (SEQ ID NO:30) VESYVPLFP, (SEQ ID NO:31)FLLYQEPVLGPVR, (SEQ ID NO:32) LNF, (SEQ ID NO:33) and MHQPPQPLPPTVMFP,(SEQ ID NO:34)

and combinations thereof.
 7. A method of down regulating 4HNE-mediatedlipid peroxidation in a cell, the method comprising contacting the cellwith a modulator selected from the group of colostrinin, a constituentpeptide thereof, an active analog thereof, and combinations thereof,wherein: the active analog is an active analog of a constituent peptideof colostrinin selected from the group of SEQ ID NO:1 through SEQ IDNO:34; the active analog comprises a peptide having an amino acidsequence with at least about 15 percent proline and having at leastabout 70 percent structural similarity to one or more constituentpeptides of colostrinin; and the active analog does not interfere withcellular uptake of redox-sensitive2′,7′-dihydro-dichlorofluorescein-diacetate.
 8. Use of a modulatorselected from the group of colostrinin, a constituent peptide thereof,an active analog thereof, and combinations thereof in the manufacture ofa medicament for: reducing 4HNE-protein adduct formation; inhibiting4HNE-mediated glutathione depletion; inhibiting 4HNE-induced activationof p53 protein; and/or inhibiting 4HNE-induced activation of c-JunNH2-terminal kinases.
 9. Use of a modulator selected from the group ofcolostrinin, a constituent peptide thereof, an active analog thereof,and combinations thereof in the manufacture of a medicament for downregulating 4HNE-mediated lipid peroxidation, wherein the active analogdoes not interfere with cellular uptake of redox-sensitive2′,7′-dihydrodichlorofluorescein-diacetate.